Name: tumorsphere derivation and treatment from primary tumor cells isolated from mouse rhabdomyosarcomas
Uploaded: 2019-09-13T13:30:00
Description: Watch this Scientific Journal Video about Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas at JoVE.com

这项工作得到了埃里森医学基金会拨款AG-NS-0843-11的支持，以及NCI癌症中心支持赠款P30CA030199授予A.S.的NIH试点补助金。

小鼠的住宿、治疗和牺牲均按照桑福德·伯纳姆·普雷比斯医学发现研究所批准的IACUC协议进行。
1. 试剂制备
<ol><li>制备100 mL的细胞分离培养基：F10培养基辅以10%马血清（HS）。</li>
	<li>制备50 mL的胶原酶 II 型溶液：在 50 mL 的细胞分离介质中溶解 1 g 胶原酶 II 型粉末（注意酶的单位，每 1 mL 培养基，因为单位数量会根据批次而变化）。将溶液与溶液加数，并储存在-20°C冷冻箱?...

<ol>
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已采用多种方法从肿瘤异质细胞群中分离和表征TPC：肿瘤克隆测定、FACS分离和肿瘤球层形成测定。肿瘤克隆测定在1971年首次被描述，用于干细胞研究，后来才应用于癌症生物学29，30。该方法是以癌症干细胞的内在特性为基础，在软胶囊培养中不受约束地扩展31。该方法自开发以来，已广泛应用于癌症研究，包括肿瘤细胞异质性研究、...

癌症是一种异质疾病;此外，同一类型的肿瘤可以在不同的患者中呈现不同的基因突变，在患者体内，肿瘤由多个细胞群组成。异质性在识别负责引发和传播癌症的细胞方面提出了挑战，但其特性对于开发有效治疗至关重要。肿瘤传播细胞（TPC）的概念，一种罕见的细胞群，有助于肿瘤的发展，已经广泛审查2。尽管TPC在多种癌症中具有特征，但识别其可靠分离的标记物仍然是几种肿瘤类型3、4、5、6的挑战。,7,8,9.因此，一种不依赖于分子标记，而是依赖于TPC功能特性（高自我更新和低附着条件下生长能力）的方法，称为肿瘤层形成测定，可以广泛应用于从大多数肿瘤的TPC的识别。重要的是，这种测定也可用于扩大三氯图C，从而直接应用于抗癌药物筛选和研究抗癌1，10。
横纹肌瘤（RMS）是一种罕见的软组织肉瘤，在幼儿11中最为常见。Althoug RMS可以通过对造血标记物表达的评估进行组织学鉴定，由于肿瘤发育刺激的多种肿瘤亚型和高异质性，原源RMS细胞尚未被单体特征化。事实上，最近的研究已经产生了关于RMS是致幻或非造血源的重要科学讨论，这表明RMS可能来自不同的细胞类型，这取决于上下文12，13。14,15,16,17.对RMS细胞系进行了大量研究，利用肿瘤层形成测定来鉴定肿瘤发育的通路，并鉴定与高度自我更新人群相关的标记物的表征18,19,20,21.
然而，尽管肿瘤圈形成测定具有识别原源RMS细胞的潜力，但一种可用于原发RMS细胞的可靠方法尚未得到描述。在此背景下，我们小组最近的一项研究采用了优化的肿瘤球形成测定，用于鉴定Duchenne肌肉萎缩症（DMD）小鼠模型22中的原源RMS细胞。从肌肉组织分离出的多种肿瘤前细胞类型，测试其在低附着条件下生长的能力，从而能够将肌肉干细胞识别为营养不良环境中RMS的原源细胞。这里描述的是肿瘤层形成测定的可重复和可靠的协议（图1），它已被成功地用于识别负责小鼠RMS发育的极其罕见的细胞群。

<table>
	<tbody>
		<tr>
			<td>Accutase cell dissociation reagent</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>Detach adherent cells and dissociate tumorspheres</td>
		</tr>
		<tr>
			<td>Celigo</td>
			<td>Nexcelom</td>
			<td>Celigo</td>
			<td>Microwell plate based image cytometer for adherent and suspension cells</td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Life Technologies</td>
			<td>17101015</td>
			<td>Tissue digestion enzyme</td>
		</tr>
		<tr>
			<td>Dispase II, protease</td>
			<td>Life Technologies</td>
			<td>17105041</td>
			<td>Tissue digestion enzyme</td>
		</tr>
		<tr>
			<td>DMEM high glucose media</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>Component of tumor cells media</td>
		</tr>
		<tr>
			<td>DMEM/F12 Media</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Component of tumosphere media</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher</td>
			<td>S312500</td>
			<td>Component of FACS buffer</td>
		</tr>
		<tr>
			<td>EGF recombinant mouse protein</td>
			<td>Gibco</td>
			<td>PMG8041</td>
			<td>Component of tumosphere media</td>
		</tr>
		<tr>
			<td>FACSAria II Flow Cytometry</td>
			<td>BD Biosciences</td>
			<td>650033</td>
			<td>Fluorescent activated cell sorter</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Omega Scientific</td>
			<td>FB-11</td>
			<td>Component of tumor cells media</td>
		</tr>
		<tr>
			<td>Fluriso (Isofluornae) anesthetic agent</td>
			<td>MWI Vet Supply</td>
			<td>502017</td>
			<td>Anesthetic reagent for animals</td>
		</tr>
		<tr>
			<td>FxCycle Violet Stain</td>
			<td>Life Technologies</td>
			<td>F10347</td>
			<td>Discriminate live and dead cells</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Life Technologies</td>
			<td>16210072</td>
			<td>Component of FACS buffer</td>
		</tr>
		<tr>
			<td>Ham&#39;s F10 Media</td>
			<td>Life Technologies</td>
			<td>11550043</td>
			<td>Component of FACS buffer</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Life Technologies</td>
			<td>16050114</td>
			<td>Component of cell isolation media</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 transfection reagent</td>
			<td>ThermoFisher</td>
			<td>L3000015</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>CB40234</td>
			<td>Provides support to trasplanted cells</td>
		</tr>
		<tr>
			<td>N-2 Supplemtns (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>Component of tumosphere media</td>
		</tr>
		<tr>
			<td>Neomycin-Polymyxin B Sulfates-Bacitracin Zinc Ophthalmic Ointment</td>
			<td>MWI Vet Supply</td>
			<td>701008</td>
			<td>Eyes ointment</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Component of FACS buffer and used for washing cells</td>
		</tr>
		<tr>
			<td>pEGFP-C1</td>
			<td>Addgene</td>
			<td>6084-1</td>
			<td>GFP plasmid</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomyocin</td>
			<td>Life Technologies</td>
			<td>15140163</td>
			<td>Component of tumosphere and tumor cells media</td>
		</tr>
		<tr>
			<td>Recombinant Human &#946;FGF-basic</td>
			<td>Peprotech</td>
			<td>10018B</td>
			<td>Component of tumosphere media</td>
		</tr>
		<tr>
			<td>Recombinant mouse Flt-3 Ligand Protein</td>
			<td>R&#38;D Systems</td>
			<td>427-FL-005</td>
			<td>Recombinant protein</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFisher</td>
			<td>15250061</td>
			<td>Discriminate live and dead cells</td>
		</tr>
	</tbody>
</table>

tumorsphere derivation and treatment from primary tumor cells isolated from mouse rhabdomyosarcomas

横纹肌瘤（RMS）是儿童最常见的软组织肉瘤。尽管已作出重大努力，能够识别与 RMS 相关的常见突变，并允许对不同 RMS 亚型进行区分，但开发新疗法以进一步改善预后仍面临重大挑战。虽然通过表达的致词标记，RMS是有致血的还是非造血的，由于对起源细胞的了解仍然不足，因此仍然存在重大争议。本研究为小鼠RMS的肿瘤球测定提供了可靠的方法。该测定基于肿瘤细胞的功能特性，允许识别肿瘤中具有肿瘤生成功能的稀有种群。还介绍了测试重组蛋白、将转染方案与肿瘤球测定相结合以及评估参与肿瘤发育和生长的候选基因的程序。进一步描述是一种将肿瘤球移植到受体小鼠中以验证体内肿瘤生成功能的程序。总体而言，所述方法允许可靠地识别和测试可在不同环境中产生的稀有 RMS 肿瘤群。最后，该协议可作为药物筛选和治疗学未来发展的平台。

肿瘤球检测 细胞分离得到优化，以获得肿瘤组织中细胞群的最大异质性。首先，由于分离组织呈现形态上不同的区域，为了增加分离均匀稀有细胞群的机会，从肿瘤的多个区域进行了取样（图1A，左侧第一面板）。第二，对采集的样品进行机械解离，同时保持切碎组织大小的均质性，尽管样品中可能存在不同的电阻（?...

Watch this Scientific Journal Video about Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas at JoVE.com

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

该协议描述了一种从肿瘤层培养开始分离小鼠横纹肌瘤原发细胞、肿瘤球的形成和治疗以及异体移植的可重复方法。

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Rhabdomyosarcoma 是一种罕见的软组织肉瘤形式，以组织学特征为组织形态和 myogenic 标记的表达。然而，鉴于这些肿瘤的不同亚型的存在，以及促成其形成的刺激的异质性，确定原生细胞是非常具有挑战性的。在这里，我们描述了从从小鼠新鲜收获的肿瘤组织开始鉴定罗巴多米瘤起源细胞的可重复和可靠的方法，这种测定是肿瘤球形成测定。肿瘤球形成分析的主要优点是，它依赖于已知存在于肿瘤原细胞中的细胞特性，它也可用于特定细胞群体的扩张和富集。此外，其球体结构模仿肿瘤环境，因此可用作药物筛选研究。这种方法有可能应用于其他类型的肿瘤，因为它不需要事先了解分子标记，但它可能需要优化培养条件。肿瘤球可能需要一些时间才能形成，特别是如果试图识别肿瘤内罕见的细胞群体。因此，不建议每天看你的亚文化板，因为它可能会对你的实验结果产生负面影响。首先在37摄氏度的培养箱中用5毫升隔离介质加热10厘米的板。重500至1000毫克的肿瘤组织，并放在盘子里。将带肿瘤组织的盘子带到无菌培养罩中，用剃刀刀片切碎。为了获得最佳的消化效果，请确保碎片的大小是均匀的。将切碎的组织和细胞隔离介质转移到 15 毫升离心管中。再用四毫升介质清洗盘子，然后加到管子里。每毫升胶原酶溶液加入700单位，在37摄氏度的摇水浴中孵育1个半小时。孵育后，在室温下以300倍g旋转组织5分钟。在不破坏颗粒的情况下吸升上一液，然后将颗粒重新在10毫升的第二消化溶液中，并在摇水浴中孵育30分钟以上。第二次消化后，移液器将细胞悬浮液上下移液，并通过50毫升离心管上的70微米尼龙过滤器。然后用10毫升的细胞隔离介质清洗过滤器，以300倍g旋转下组织5分钟。吸制上一提液，并在肿瘤细胞介质的20毫升中重新暂停颗粒。将悬浮液转移到15厘米培养板，并一夜之间将细胞放在37摄氏度的培养箱中。第二天，改变介质以清除可能对细胞生存产生负面影响的碎片和死细胞。此时，评估细胞汇合，让细胞在培养箱中生长，直到它达到90%，每天监测细胞，每两天更换一次培养。对于肿瘤球衍生，使用通道P1或P2的细胞，以避免细胞通过多个通道选择。用 PBS 清洗细胞盘，然后用分离溶液盖住它们。将它们放在培养箱中5至10分钟，然后在明亮的显微镜下观察盘子，确认分离。一旦细胞分离，将肿瘤细胞介质添加到板中，并将细胞悬浮液转移到离心管。将细胞以300倍g旋转5分钟，去除上心，并在肿瘤球介质中重新暂停细胞。重新发送细胞后，使用 Trypan 蓝色计算细胞并计算细胞浓度。将适当数量的细胞板在96井的低附板中，将细胞放在培养箱中，直到实验结束，除非补充介质，否则注意不要打扰板。实验完成后，在明亮的领域显微镜下或使用Celigo软件手动识别肿瘤球。肿瘤球的数量和大小可以通过这种测定进行评估。为了准备肿瘤球进行全血移植，请将从特定细胞类型或治疗中获得的所有肿瘤球汇集在一起。离心肿瘤球，小心地用一毫米移液器取出上流液，然后用200微升移液器清洗，然后用10毫升无菌PBS清洗。再次旋转肿瘤球并吸气 PBS。在肿瘤球颗粒顶部的一毫升细胞分离溶液中加入500微升，在37摄氏度的摇晃水浴中孵育细胞。每10分钟检查一次消化进度。整个过程最多需要 30 分钟。如果震动水浴中肿瘤球的消化不足以获得单细胞溶液，则建议通过上下移液进行机械干扰。请注意，旋转肿瘤球后不会形成稳定的颗粒，因此应用一毫升移液器和200微升移液器小心吸升。获得单细胞溶液后，加入一卷肿瘤细胞介质，以300倍g旋转，在4摄氏度下旋转5分钟。离心后，所有后续步骤都必须在冰上执行。在冷肿瘤细胞培养中重新发送细胞，并使用 Trypan 蓝色排除计数活细胞。确定正常数量的细胞，并在50微升的冷肿瘤细胞介质中稀释。将移液器尖端放在冷肿瘤细胞介质中冷却，然后用它来服用 50 微升的冷 ECM 溶液，然后用细胞将其添加到管中，确保在这个过程中不要从冰中去除 ECM 管。在移植之前将细胞保持在冰上，并在冰上放置一个带盖的0.5毫升胰岛素注射器，并放置一个29量针。在确认小鼠已正确麻醉后，剃掉动物的右侧，将细胞溶液吸入冷却的注射器中，然后将细胞皮下注射到剃光区域。如果注射正确执行，皮肤下将形成可见的凸起。该协议可用于可靠地形成肿瘤球，用于识别负责软组织肉瘤发育的稀有细胞群。这种测定的一个基本部分是肿瘤球和细胞群之间的区分，它们表现出明显的形态差异。为了确定感兴趣的蛋白质对肿瘤细胞产生影响的最佳浓度，有必要评估蛋白质下游靶点表达水平。三个测试的基因显示对重组蛋白治疗有剂量依赖性反应，1个没有。为了建立有效的方案，分析了转染对固有肿瘤细胞的影响;对两种不同量的试剂进行了检测，用GFP-报告粒评估了效率。减少试剂量可提高转染效率。必须实施两步协议，对悬浮细胞进行转染。在粘附细胞上进行转染，24小时后分离并悬浮镀上;7天后，受控肿瘤球表达GFP。肿瘤球获得、治疗和移植后，可以比较这种不同治疗对体内肿瘤生长的影响。这些方法将使科学家能够回答Rhabdomyosarcomas细胞的仍然存在的问题，同时为药物筛查确定基础。

tumorsphere-derivation-treatment-from-primary-tumor-cells-isolated

Research

JoVE Journal

Genetics

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

从天然产物其生物合成基因簇：一个演示使用Polyketomycin从<em&gt;链diastatochromogenes</em&gt;Tü6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

科研

遗传学

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

原发性人类 CD4+ T 淋巴细胞低线粒体 DNA 污染的 ATAC-seq 检测

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...